The wavelength position and structure of the fluorescence emission spectrum of a fluorescent sample in the gaseous phase or in liquid respectively solid solution is typical of the chemical composition of the sample and is also typical of the sample phase. Fluorescence emission has a nanosecond duration and upon excitation of the fluorescence of a sample by a subnanosecond radiation pulse the fluorescence emission intensity decay curve can be recorded as a function of time. This decay curve is again typical of the chemical composition and the phase of the sample.
The fluorescence emission intensity originating from a fluorescent substance is linearly proportional to the concentration of the substance and in known apparatus the fluorescence emission intensity is measured in order to determine the fluorescent substance concentration in the sample. Often, in cases where the nature of an unknown substance has to be determined, the fluorrescence wavelength spectrum is recorded. As shown in FIG. 6, such measurements usually are carried out by illuminating the sample with light from a continuous light source, such as a high-pressure xenon arc, or a pulsed light source, such as a flash lamp, the excitation radiation being passed through a monochromator (A) selecting the excitation wavelength prior to falling into the sample and the resulting fluorescence emission being passed through a monochromator (B) prior to falling onto a fluorescence radiation detector. The wavelength settings of the two monochromators are ideally such that the light passing through monochromator (A) cannot pass through monochromator (B), in this way avoiding that the radiation detector measuring the fluorescence emission intensity will also be irradiated with light originating from the excitation light source.
However, even the best monochromators have a finite transmission for other wavelengths but the central setting wavelength in the form of stray light and the sensitivity of known fluorescence spectrophotometric apparatus is primarily limited by the circumstance that all fluorescent samples, apart from absorbing the excitation radiation causing the sample fluorescence, will scatter part of the excitation radiation and as monochromator (B) will transmit part of this scattered excitation radiation independently of its central wavelength setting, scattered radiation will be able to reach the fluorescence radiation detector. When the concentration of the fluorescent substance in the sample is so low that its fluorescence intensity is less than the intensity of the scattered excitation radiation reaching the detector, the fluorescence emission cannot be detected in known apparatus.
The light scattering ability of molecules increases with increasing molecular size and consequently the study of the fluorescence emission from biological systems, such as proteines and living tissues, is seriously hampered by light scattering. However, fluorescence studies of such systems are of central importance in the elucidation of biochemical aspects of human diseases and the object of the present invention is to provide a method and apparatus which overcome the sensitivity limitations of known fluorescence spectrophotometers and thus provide a means of analysing hitherto undetectable low levels of fluorescence intensity.